From the Cardiovascular Research Institute and Diabetes Obesity and Metabolism Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York 10029.

2

Department of Pharmaceutical Science, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064-3095.

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Departments of Medicine and Neuroscience and Diabetes Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, and.

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Celladon Corporation, San Diego, California 92130-3579.

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From the Cardiovascular Research Institute and Diabetes Obesity and Metabolism Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York 10029, djamel.lebeche@mssm.edu.

Abstract

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.

CDN1163 increases glucose tolerance in ob/ob mice.A, intraperitoneal glucose tolerance test (GTT) assessed on day 8 in lean, vehicle-treated obese (Ob) and CDN1163-treated obese (Ob+CDN) mice (n = 10/group); glucose was measured at times shown after 1 g/kg of glucose injection and calculated as the area under the curve (AUC). B, the intraperitoneal insulin tolerance test was assessed on day 11 after 1 IU/kg of insulin injection 2 h after CDN injection, and glucose clearance is expressed as % reduction from basal levels, and (B) plasma insulin levels at end of study (day 50) are shown for lean and vehicle- and CDN1163-treated obese mice (n = 10) (C). D, representative from at least three experiments of Western blot analysis of insulin-stimulated Akt phosphorylation at serine 473 (pS-Akt) and threonine 308 (pT-Akt) in the liver after an acute insulin bolus (36.3 μg/ml in 0.9% saline (1 units/kg, assuming potency of 27.5 units/mg)). Protein loading was verified with total Akt and GAPDH. Also shown is Western blot liver SERCA2b expression in the three different groups of mice with GAPDH as a loading control. E, quantitative real-time PCR analysis of genes involved in gluconeogenesis in the liver in vehicle-treated obese (Ob) and CDN1163-treated (Ob+CDN) mice. Data are expressed as the means ± S.E. from at least 3–5 determinations. *, p < 0.05; **, p < 0.01; ***, p < 0.001, lean versus obese; #, p < 0.05 and ###, p < 0.001, obese versus CDN1163-treated obese mice. Data in C–E were performed at the end of the study (i.e. day 50). G6Pase, glucose-6-phosphatase.

Schematic diagram depicting proposed molecular mechanism underlying CDN1163/SERCA2b metabolic benefits. A fundamental abnormality of obesity and diabetes is down-regulation and dysfunction of Serca2b causing intracellular Ca2+ imbalance with a concomitant induction of ER stress and mitochondrial dysfunction, as these two events are reciprocally related. ER stress and mitochondrial dysfunction then trigger insulin resistance and development of diabetes. Pharmacological restoration of Serca2b activity by CDN1163 (a) normalizes intracellular Ca2+ dyshomeostasis (which reestablishes ER homeostasis) and (b) activates AMPK, which in turn up-regulates PGC1α (which improves mitochondrial biogenesis) and down-regulates HNF4α leading to suppression of gluconeogenesis. Evidence also shows that AMPK can improve ER stress and Ca2+ and has long played a major role in mitochondria (dashed lines). Attenuation of gluconeogenesis and ER stress and improvement of mitochondrial efficiency altogether then ameliorate insulin resistance and diabetes.